The patch clamp technique [references 1, 2] is the gold standard in neuroscience, cell biology, and pharmacology to study the electrophysiological properties of membrane-embedded proteins that regulate the ionic permeability of the plasma membrane-ion channels, receptors, and transporters. Changes in membrane-permeability result in ionic membrane currents and respective changes in transmembrane voltage. “Patch clamp” refers to recording these electrical signals from a membrane-patch (or entire membrane in whole-cell) using electronic circuitry which clamps either the membrane current or the transmembrane voltage to a desired value or a desired time course. In combination with patch-clamping, investigators also apply and record other signals such as chemical, mechanical, thermal, magnetic, and electromagnetic waves. The analysis of recorded stimulus-response data sets allows one to infer the roles of membrane-embedded proteins in the macro and microscopic phenomena involved in intra- or intercellular communication.
In a conventional whole-cell patch-clamp experiment, a pipette containing an electrolyte solution is used to gain electrical access to the interior of the cell (FIG. 1). A stationary Ag/AgCl wire in the pipette contacts the solution and connects to the amplifier input. The pipette is advanced towards the cell until its tip touches the plasma membrane. Gentle suction then applied inside the pipette seals its tip with the cell membrane. The seal is called a “Gigaohm seal” or a “Gigaseal” as its electrical resistance must exceed several GΩ to allow reliable recordings. The pipette-cell configuration in this instance is called “cell-attached”. In order to obtain whole-cell configuration, a somewhat larger amount of suction is applied inside the pipette than was required for forming the seal, thereby rupturing the cell membrane giving the amplifier “low” (kΩ ohms or MΩ) resistance electrical access to the interior of the cell. In an alternative, less common, method of achieving the whole-cell configuration, a voltage pulse usually called “zap” is applied to breakdown the lipid bilayer, though this technique does not always work for all cell types.
Although widely used, the conventional whole-cell patch-clamp experiment is laborious and has a low yield owing to the following problems.
1. It is difficult to form and maintain gigaseals and obtain whole-cell configuration from cell-attached mode. The experimenter has to practice and gain experience in order to do so. In case of a failure to obtain whole-cell configuration with one pipette, the experimenter has to replace it with a new one. Even experienced experimenters often have to try a couple of pipettes before they achieve whole-cell configuration. Currently, there are no alternative methods other than those mentioned above to obtain whole-cell configuration in patch clamp experiments.
2. The presence of series (or access) resistance results in voltage clamp errors and limits the bandwidth of the recorded signals. The bulk of series resistance is located at the pipette tip, the narrowest region of the conduction channel. This resistance sometimes increases during an experiment when cellular components migrate to and reduce the conduction channel [3]. To counter these problems, patch clamp amplifiers and data analysis software provide ways to compensate for series resistance [4], [5], [6]. However, compensation can be inadequate while recording high frequency signals (such as gating currents) [7] or while recording from small neural structures [8] such as dendrites or axons.
3. In some experiments, the diffusion of cytosolic chemicals into the pipette or components of the pipette solution into the cytosol, can dilute soluble components of the cytoplasm needed for ion channel function thereby resulting in erroneous results [9]. One approach to overcoming this problem is to reduce the pipette orifice diameter limiting diffusion but at the cost of an increase in series resistance. When this approach is not viable, cytosolic extract can be added to the pipette solution to minimize ionic concentration gradients reducing diffusion. The “perforated patch configuration” is an alternative from cell-attached mode where the membrane perforation is attained with ionophores (e.g. ATP, polyene antibiotics) [10]. The later two methods are laborious and difficult to carry out.
The conventional patch clamp technique has resulted in a large number of productive studies. However, there remains room to improve the patch clamp electrode to provide a coherent solution to the problems mentioned above.